Gelshift Chemiluminescent EMSA Method

The Gelshift Chemiluminescent EMSA Kit provides a non-radioactive method to detect DNA-protein interactions. In this method, cell extracts or purified factor are incubated with a biotin 3' or 5' end-labeled DNA probe containing the consensus binding site of interest. Samples are then resolved by electrophoresis on a native polyacrylamide gel and transferred to a nylon membrane. The biotin end-labeled DNA probe is detected using streptavidin conjugated to horseradish peroxidase (HRP) and a chemiluminescent substrate. Samples in which the protein of interest bound the target DNA will migrate slower than DNA alone resulting in a "shift" of the labeled DNA band.

Example EMSA Binding Reactions

Biotin-labeled DNA = no shift in the absence of cell extract or purified factor

Biotin-labeled DNA + Extract = DNA shift due to the binding of protein to the labled DNA probe

Biotin-labeled DNA + Extract + Excess Unlabeled DNA = no shift as the excess of unlabeled DNA competes for binding of the target protein in the extract; this reaction verifies the specificity of the protein-DNA interaction

Gelshift Chemiluminescent EMSA Advantages

Non-radioactive assay

Better sensitivity than radioactive or digoxigenin methods

Fast procedure can be completed in 5 hours

Additional reagents supplied to help optimize conditions for your sample system

Includes control DNA and extract to help new users understand the methods as they optimize conditions for their sample systems

Gelshift or Electrophoretic Mobility Shift (EMSA) Info

Electrophoretic mobility shift assays (EMSA), also known as gel shifts, gel retardation assays or mobility assays can be used to study DNA-protein interactions1-3. The principle behind EMSA relies on the fact that DNA-protein complexes migrate slower than DNA alone in a native polyacrylamide or agarose gel. This difference in electrophoretic separation of DNA-protein complexes can be visualized as a "shift" in migration of the labeled DNA band. This enables researches to screen different nuclear extracts or gene promoters for specific transcription factor DNA binding activity.

Better sensitivity

The non-radioactive format of the Gelshift Chemiluminescent EMSA Assay Kit does not sacrifice sensitivity when compared to 32P or digoxigenin methods.

HeLa nuclear extract (6.8 µg) was incubated with 20 fmol Oct-1 duplex DNA labeled for use in either the Gelshift Chemiluminescent EMSA kit, a digoxigenin-based EMSA or with 32P using T4 polynucleotide kinase (40,000 cpm/reaction) for use in a radioactive EMSA. The kits were used according to the manufacturer's instructions. The radioactive EMSA was exposed directly to X-ray film using screens.

The Gelshift Chemiluminescent EMSA Kit was used to obtain sensitive gel shift results for four different DNA-protein complexes. Biotin-labeled target duplexes were incubated with HeLa nuclear extract (Oct-1, AP1 and NFκB) or the provided Control Nuclear Extract from the kit. Unlabeled competitor sequences were used at 200-fold molar excess over labeled DNA. The Control reactions from the Epstein-Barr Nuclear Antigen (EBNA) system were supplemented with 2.5% glycerol and 0.05% NP-40, while the AP1 reactions were supplemented with 10% glycerol. X-ray film exposure time varied from 2 minutes for the EBNA Control, Oct-1 and AP1 to 5 minutes for NFκB.

References

Active Motif's Supershift and Gel shift Buffers have been shown to significantly reduce non-specific background. These buffers are available for purchase to save you the time and inconvenience of optimizing buffers for your own experiments.

Binding Buffers B-1 through B-4 are for incubation of the extract:antibody mixture, while Binding Buffers C-1 through C-3 facilitate binding of the labeled probe.